Purpose: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo.

Methods: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting.

Results: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization.

f2: Olomoucine treatment does not disrupt the integrity of the epithelial cell sheet. A: The immunofluorescence of E-cadherin in epithelial cells of corneal whole mounts of control animals shows tightly packed epithelial cells with a smooth, advancing cell front. Cells were compact and cell density was high especially along the wound edge. B: Higher magnification of the boxed area shown in panel A shows that E-cadherin immunofluorescence in control corneas was confined almost entirely to cell-to-cell boundaries. Punctate E-cadherin staining was seen at the migrating front on the edge lacking cell-to-cell contacts (arrowhead). C: Superimposition of E-cadherin immunostaining and DAPI-staining of nuclei confirms that E-cadherin is located at cell-to-cell boundaries and that all cells express E-cadherin. D: In olomoucine-treated corneas, cell density was appreciably lower and the migrating front was irregular. Cell-to-cell junctions appeared to be intact except for a few cells at the wound edge. Immunostaining of E-cadherin was weak or undetectable at the migrating front on the edge lacking cell-to-cell contacts (arrowhead). E: Higher magnification of the boxed area shown in panel D demonstrates punctate intracellular immunostaining for E-cadherin in many cells (single arrows). E-cadherin localization at cell-to-cell junctions was disrupted in a few cells along the wound edge (double arrows). F: Superimposition of E-cadherin immunofluorescence and DAPI-staining of nuclei confirms that E-cadherin is located at cell-to-cell boundaries and demonstrates that all cells express E-cadherin. Scale bar=100 μM in A,C,D, F; 40 μM in B,E.

Mentions:
Since previous studies had suggested that inhibition of Cdk5 activity might also affect E-cadherin junctional stability, we examined E-cadherin localization in treated and untreated corneas 18 h after wounding. In control corneas, E-cadherin immunostaining was confined almost entirely to cell-cell boundaries (Figure 2). Only cells at the leading edge, which lacked cell contacts on one surface, showed appreciable cytoplasmic staining of E-cadherin. Cells were compact and cell density was high especially along the wound edge. In contrast, in olomoucine-treated corneas, cells were more spread and cell density was appreciably lower. Cell-cell junctions appeared to be intact for the most part, although many cells contained E-cadherin positive vesicles (Figure 2). Small disruptions in the migrating front suggest there may be some loss of cell-cell adhesion in this region, but it is not widespread.

f2: Olomoucine treatment does not disrupt the integrity of the epithelial cell sheet. A: The immunofluorescence of E-cadherin in epithelial cells of corneal whole mounts of control animals shows tightly packed epithelial cells with a smooth, advancing cell front. Cells were compact and cell density was high especially along the wound edge. B: Higher magnification of the boxed area shown in panel A shows that E-cadherin immunofluorescence in control corneas was confined almost entirely to cell-to-cell boundaries. Punctate E-cadherin staining was seen at the migrating front on the edge lacking cell-to-cell contacts (arrowhead). C: Superimposition of E-cadherin immunostaining and DAPI-staining of nuclei confirms that E-cadherin is located at cell-to-cell boundaries and that all cells express E-cadherin. D: In olomoucine-treated corneas, cell density was appreciably lower and the migrating front was irregular. Cell-to-cell junctions appeared to be intact except for a few cells at the wound edge. Immunostaining of E-cadherin was weak or undetectable at the migrating front on the edge lacking cell-to-cell contacts (arrowhead). E: Higher magnification of the boxed area shown in panel D demonstrates punctate intracellular immunostaining for E-cadherin in many cells (single arrows). E-cadherin localization at cell-to-cell junctions was disrupted in a few cells along the wound edge (double arrows). F: Superimposition of E-cadherin immunofluorescence and DAPI-staining of nuclei confirms that E-cadherin is located at cell-to-cell boundaries and demonstrates that all cells express E-cadherin. Scale bar=100 μM in A,C,D, F; 40 μM in B,E.

Mentions:
Since previous studies had suggested that inhibition of Cdk5 activity might also affect E-cadherin junctional stability, we examined E-cadherin localization in treated and untreated corneas 18 h after wounding. In control corneas, E-cadherin immunostaining was confined almost entirely to cell-cell boundaries (Figure 2). Only cells at the leading edge, which lacked cell contacts on one surface, showed appreciable cytoplasmic staining of E-cadherin. Cells were compact and cell density was high especially along the wound edge. In contrast, in olomoucine-treated corneas, cells were more spread and cell density was appreciably lower. Cell-cell junctions appeared to be intact for the most part, although many cells contained E-cadherin positive vesicles (Figure 2). Small disruptions in the migrating front suggest there may be some loss of cell-cell adhesion in this region, but it is not widespread.

Bottom Line:
Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro.Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05).The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization.

Purpose: To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo.

Methods: Corneal debridement wounds of 1.5 mm were made on the ocular surface of CD-1 mice. A 20 microl drop of 15 microM olomoucine in 1% DMSO was applied to the wound area immediately after wounding and again after 6 h. Control mice received identical applications of 1% DMSO. Mice were euthanized after 18 h, two weeks, and three weeks for evaluation of wound healing and restratification. Corneas were stained with Richardson's dye, photographed, and processed for histology and immunofluorescence as whole mounts or paraffin sections. The remaining wound area at 18 h was measured by image analysis. Scratch wounded cultures of human corneal-limbal epithelial cells (HCLE) were used to examine the effect of olomoucine on matrix metalloproteinase (MMP) expression in vitro. MMP-2 and MMP-9 were detected by immunofluorescence and immunoblotting.

Results: Olomoucine treatment significantly enhanced corneal wound closure without increasing inflammation or infiltration of polymorphonuclear leukocytes 18 h after wounding (p<0.05). The increased localization of MMP-9 within epithelial cells at the wound edge was further enhanced by olomoucine while the expression of MMP-2 was reduced. Olomoucine treatment of scratch wounded HCLE cells produced similar changes in MMP-9 and MMP-2 expression. The examination of treated corneas two and three weeks after wounding showed normal epithelial restratification with no evidence of inflammation or stromal disorganization.